Bottom Line:
Fittingly, SUMOylation mutant AR cells proliferate faster and are more sensitive to apoptosis.Moreover, ChIP-seq analyses show that the SUMOylation can modulate the chromatin occupancy of AR on many loci in a fashion that parallels their differential androgen-regulated expression.De novo motif analyses reveal that FOXA1, C/EBP and AP-1 motifs are differentially enriched at the wtAR- and the AR-K386R,K520R-preferred genomic binding positions.

Figure 6: De novo motif analysis of the AR-binding sites (ARBs) identified in the PC-3 cells. De novo analysis was performed on ±100 bp of the AR peak center. (A) The top five motif matrices predicted by the de novo motif analysis by HOMER are shown. (B) Enrichment of de novo motifs in the wtAR-, the AR-2KR-preferred or the wtAR- and AR-2KR-shared ARBs. Statistically significant differences (***P < 0.001, **P < 0.01; Chi-square test) between wtAR and AR-2KR-preferred binding sites de novo motifs are indicated. (C) ChIP-seq track examples of the AR-binding events and the corresponding DNA sequences of the de novo motifs in SAFB, ANKRD17 and ADRA1B loci. Occupancies of FOXA1 and C/EBPβ in SAFB and ANKRD17 and c-Jun in ADRA1B locus after 1-h R1881 exposure were monitored using qChIP (bottom right hand panels). Results represent the means (n = 3) ± SDs and are shown as fold over to IgG-immunoprecipitated samples. Statistically significant differences (**P < 0.01 and *P < 0.05; Student's t-test) between the wtAR- and the AR-2KR-expressing cells are indicated.

Mentions:
We next performed de novo motif analyses with all ARBs to identify over-represented transcription factor-binding motifs in the ARBs. As expected, the de novo ARE consensus (that closely resembles the ARE motif in the JASPAR-database) was the most enriched motif among all ARBs (in >60% of wtAR-, AR-2KR-preferred and shared ARBs) (Figure 6A and B). Further analysis revealed no clear differences in the number of AREs per ARE-containing ARBs between the preferred and shared ARBs, with the means (±SD) 1.28 ± 0.52, 1.20 ± 0.47 and 1.31 ± 0.56 for the wtAR-, the AR-2KR- and the shared ARBs, respectively. Other significantly enriched motifs were those for ERG, FOXA1, C/EBP and AP-1 (Figure 6A). While the de novo ARE and ERG motifs were fairly similarly enriched in all three ARB subsets, there were significant differences in the enrichment of FOXA1, C/EBP and AP-1 motifs to the three ARB subsets. Based on western blotting analysis, factors binding to these motifs (FOXA1, C/EBPβ and c-Jun, major AP-1 component) are expressed in our PC3 model cells (Supplementary Figure S10). Both FOXA1 and C/EBP motifs were found three times more often within the wtAR-preferred then AR-2KR-preferred ARBs, whereas the AP-1 motif was nearly three times more prevalent within the AR-2KR-preferred ARBs (Figure 6B and C, Supplementary Figure S11). As shown in Figure 6C, more pronounced loading of both the FOXA1 and the C/EBPβ, for example, onto SAFB and ANKRD17 loci in wtAR than AR-2KR cells is in line with the in silico predictions in panel B. Moreover, c-Jun appears to be more avidly loaded, for example, onto ADRA1B locus in AR-2KR- than in wtAR-expressing PC3 cells (Figure 6C). De novo analysis of the HEK293 data showed, in addition to the AREs, a similar over-enrichment of FOXA1 within the wtAR-preferred ARBs. GATA1 was also over-enriched in the wtAR-preferred ARBs, whereas EBF1 and PAX2 were more prevalent in the AR-2KR preferred ARBs (Supplementary Figure S12A and B). The differences in the enriched motifs between the PC-3 and the HEK293 cells likely reflect cell-specific differences in the expression of their cognate transcription factors. Compared to LNCaP and VCaP prostate cancer cells in which about half of the identified ARBs have a FOXA1 motif (31,32), the PC-3 ARBs and the HEK293 ARBs less frequently harbor a FOXA1 motif. This may be due to the lower amount of FOXA1 in the latter two cell lines (Supplementary Figure S10).

Figure 6: De novo motif analysis of the AR-binding sites (ARBs) identified in the PC-3 cells. De novo analysis was performed on ±100 bp of the AR peak center. (A) The top five motif matrices predicted by the de novo motif analysis by HOMER are shown. (B) Enrichment of de novo motifs in the wtAR-, the AR-2KR-preferred or the wtAR- and AR-2KR-shared ARBs. Statistically significant differences (***P < 0.001, **P < 0.01; Chi-square test) between wtAR and AR-2KR-preferred binding sites de novo motifs are indicated. (C) ChIP-seq track examples of the AR-binding events and the corresponding DNA sequences of the de novo motifs in SAFB, ANKRD17 and ADRA1B loci. Occupancies of FOXA1 and C/EBPβ in SAFB and ANKRD17 and c-Jun in ADRA1B locus after 1-h R1881 exposure were monitored using qChIP (bottom right hand panels). Results represent the means (n = 3) ± SDs and are shown as fold over to IgG-immunoprecipitated samples. Statistically significant differences (**P < 0.01 and *P < 0.05; Student's t-test) between the wtAR- and the AR-2KR-expressing cells are indicated.

Mentions:
We next performed de novo motif analyses with all ARBs to identify over-represented transcription factor-binding motifs in the ARBs. As expected, the de novo ARE consensus (that closely resembles the ARE motif in the JASPAR-database) was the most enriched motif among all ARBs (in >60% of wtAR-, AR-2KR-preferred and shared ARBs) (Figure 6A and B). Further analysis revealed no clear differences in the number of AREs per ARE-containing ARBs between the preferred and shared ARBs, with the means (±SD) 1.28 ± 0.52, 1.20 ± 0.47 and 1.31 ± 0.56 for the wtAR-, the AR-2KR- and the shared ARBs, respectively. Other significantly enriched motifs were those for ERG, FOXA1, C/EBP and AP-1 (Figure 6A). While the de novo ARE and ERG motifs were fairly similarly enriched in all three ARB subsets, there were significant differences in the enrichment of FOXA1, C/EBP and AP-1 motifs to the three ARB subsets. Based on western blotting analysis, factors binding to these motifs (FOXA1, C/EBPβ and c-Jun, major AP-1 component) are expressed in our PC3 model cells (Supplementary Figure S10). Both FOXA1 and C/EBP motifs were found three times more often within the wtAR-preferred then AR-2KR-preferred ARBs, whereas the AP-1 motif was nearly three times more prevalent within the AR-2KR-preferred ARBs (Figure 6B and C, Supplementary Figure S11). As shown in Figure 6C, more pronounced loading of both the FOXA1 and the C/EBPβ, for example, onto SAFB and ANKRD17 loci in wtAR than AR-2KR cells is in line with the in silico predictions in panel B. Moreover, c-Jun appears to be more avidly loaded, for example, onto ADRA1B locus in AR-2KR- than in wtAR-expressing PC3 cells (Figure 6C). De novo analysis of the HEK293 data showed, in addition to the AREs, a similar over-enrichment of FOXA1 within the wtAR-preferred ARBs. GATA1 was also over-enriched in the wtAR-preferred ARBs, whereas EBF1 and PAX2 were more prevalent in the AR-2KR preferred ARBs (Supplementary Figure S12A and B). The differences in the enriched motifs between the PC-3 and the HEK293 cells likely reflect cell-specific differences in the expression of their cognate transcription factors. Compared to LNCaP and VCaP prostate cancer cells in which about half of the identified ARBs have a FOXA1 motif (31,32), the PC-3 ARBs and the HEK293 ARBs less frequently harbor a FOXA1 motif. This may be due to the lower amount of FOXA1 in the latter two cell lines (Supplementary Figure S10).

Bottom Line:
Fittingly, SUMOylation mutant AR cells proliferate faster and are more sensitive to apoptosis.Moreover, ChIP-seq analyses show that the SUMOylation can modulate the chromatin occupancy of AR on many loci in a fashion that parallels their differential androgen-regulated expression.De novo motif analyses reveal that FOXA1, C/EBP and AP-1 motifs are differentially enriched at the wtAR- and the AR-K386R,K520R-preferred genomic binding positions.